Cytochrome P450 enzymes (CYPs) in humans are a family of heme-containing enzymes involved in metabolism of steroids, vitamins, eiconsanoids, and xenobiotics such as drugs, complex plant alkaloids, carcinogens, and other small molecules. The role of CYPs in metabolism of nearly all drugs has made this family of enzymes of considerable interest in medicine and for human health. CYP2D6 is one of the significant drug metabolizing enzymes responsible for metabolism of ~25% of pharmaceutical compounds while only accounting for a fraction of liver CYP content. The enzyme also displays multiple polymorphic forms that contribute to inter-individual difference in responses to drugs metabolized by CYP2D6. One caveat of such broad substrate specificity among drug metabolizing CYPs is that the enzymes are susceptible to inhibition by products of their own reactions. This type of inhibition is called mechanism-based inactivation and leads to irreversible inactivation of the enzyme. Compounds that act as mechanism-based inactivators are important tools for understanding structure/function of these enzymes and there are only a few known for CYP2D6. The project proposed here will contribute to understanding mechanisms inactivation of CYP2D6 and select allelic variants by two classes of inactivators - protein adductors and heme modifiers. These complimentary lines of investigation will be pursued through two aims: Aim 1: To determine the amino acid(s) of CYP2D6 targeted by mechanism-based protein adduct inactivators. In silico and LC/MS predictions of amino acids involved in apoprotein adduction will be tested by mutagenesis of postulated nucleophiles to non-nucleophilic amino acids. The expressed mutant enzyme(s) will be characterized in terms of kinetics with normal and inactivator substrates including two protein adductors (SCH66712 and EMTPP) and two heme modifiers (paroxetine and MDMA). Aim 2: To determine the interaction of the most common polymorphisms of CYP2D6 with inactivators. Almost 20% of drug therapies are affected by polymorphisms. Polymorphisms lead to different plasma concentrations and half-life of drugs that lead to adverse drug reactions. CYP2D6 is highly polymorphic and the role of polymorphisms in inactivation will be characterized. Together, these studies will provide an understanding of how in activators interact with CYP2D6 and lead to new opportunities in rational drug design and evaluation of inter-individual differences in drug metabolism in the age of personalized medicine.